In this second renewal we propose to pursue three new areas of investigation that are a direct consequence of advances made during the previous funding period. In Aims 1 and 2 we will further define the role that SHIP plays in NK effector function with a focus on cytokine production and NK tolerance to host. In Aim 3 we will capitalize on our recent identification of the first small molecules that can selectively target SHIP1 activity in vivo to determine if this novel compound can enhance cancer control by NK cells. The specific aims are: Aim 1: Determine the molecular basis for defective cytokine production by SHIP-/- NK cells. During the previous funding period we made the unexpected observation that SHIP is required for effective induction of 3-IFN by major NK activating receptors like NK1.1, NCR1/NKp46 and NKG2D. In this aim we will determine how broadly SHIP- deficiency impairs production of 3-IFN and other cytokines and chemokines produced by NK cells. In addition, we will define the molecular basis of this defect. Aim 2: Define the role of SHIP in self tolerance by the NK cell compartment. During the previous funding period we found that the expression and representation of both murine (Ly49) and human (KIR) NK inhibitory receptors for MHC class I ligands is limited by SHIP and that SHIP preferentially plays this role in the context of high affinity MHC-I ligands for these receptors. In the case of human KIR, we provide preliminary evidence that SHIP is required to maintain NK tolerance to the host. In this aim we will determine if SHIP plays a role in NK tolerance to self and does so in the context of MHC-I ligands that vary in their affinity for NK inhibitory receptors. Aim 3: Test the potential of SHIP inhibitors to enhance NK cell eradication of cancer. During the previous funding period we developed the first small molecule inhibitors of SHIP enzyme activity, one of which is selective for SHIP1 and has significant activity in vivo. In addition, we demonstrated that SHIP-/- NK cells from an MHC haplotype with a high affinity MHC-I ligand demonstrate supernormal killing of allogeneic tumors that lack this ligand. SHIP is also known to limit ADCC against tumor cells by NK cells. Thus, in this aim we will determine if chemical inhibition of SHIP can improve the ability of NK cells to eradicate cancer in vivo. This will be examined in three different contexts each of which has significant potential for translation to clinical therapy.